Synthesis, DNA binding and antitrypanosomal activity of benzimidazole analogues of DAPI

Article abstract of DOI:10.1016/j.bmcl.2016.11.006

A series of novel benzimidazole diamidines were prepared from the corresponding dicyano analogues either by applying Pinner methodology (5a–c, 10 and 13a) or by making amidoximes intermediates that were reduced to the corresponding amidines (15a–c). The n

Full text of DOI:10.1016/j.bmcl.2016.11.006

Accepted Manuscript
Synthesis, DNA binding and Antitrypanosomal Activity of Benzimidazole An-
alogues of DAPI
Abdelbasset A. Farahat, Cheree Bennett-Vaughn, Ekaterina M. Mineva, Arvind
Kumar, Tanja Wenzler, Reto Brun, Yang Liu, W. David Wilson, David W.
Boykin
PII:
S0960-894X(16)31146-5
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.11.006
BMCL 24401
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
27 September 2016
31 October 2016
1 November 2016
Please cite this article as: Farahat, A.A., Bennett-Vaughn, C., Mineva, E.M., Kumar, A., Wenzler, T., Brun, R., Liu,
Y., David Wilson, W., Boykin, D.W., Synthesis, DNA binding and Antitrypanosomal Activity of Benzimidazole
Analogues of DAPI, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.
2016.11.006
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Synthesis, DNA binding and Antitrypanosomal Activity of Benzimidazole
Analogues of DAPI
Abdelbasset A. Farahata,b *, Cheree Bennett-Vaughna, Ekaterina M. Minevaa,
Arvind Kumara, Tanja Wenzlerc,d, Reto Brunc,d, Yang Liua, W. David
Wilsona and David W. Boykina.
aDepartment of Chemistry, Georgia State University, Atlanta, GA, 30303.
bDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Mansoura University, Mansoura 35516, Egypt.
cSwiss Tropical and Public Health Institute, Basel, 4002, Switzerland,
dUniversity of Basel, Basel, 4003, Switzerland
* Fax: 0014044135505, E-mail: afarahat@gsu.edu
Abstract
A series of novel benzimidazole diamidines were prepared from the
corresponding dicyano analogues either by applying Pinner methodology (5
a-c, 10 and 13 a) or by making amidoximes intermediates that were reduced
to the corresponding amidines (15 a-c). The new amidines were evaluated in
vitro against the protozoan parasite Trypanosoma brucei rhodesiense (T. b.
r.). The thiophene analogue 5b and the N-methyl compound 15a showed
superior antitrypanosomal activity compared to that of the parent I.
Keywords. Benzimidazole diamidines, DAPI, DNA minor groove binders,
antitrypanosomal activity.
1

Benzimidazole analogues have been extensively studied as AT specific
1
DNA minor groove binders . Benzimidazole containing drugs have been
2
3
4
used as analgesic, anti-inflammatory , antihistaminic , anthelmintic ,
5
6
antiulcer , and antiamoebic
agents. Also, benzimidazole containing
compounds have shown good antiprotozoal activity7b,c. It has been estimated
that over 50 million people are at risk for contracting Human African
Trypanosomiasis (HAT) in more than 30 African countries and there
remains a clear need to develop new safe, efficacious and affordable drugs 8.
Over sixty years ago, 5-amidino-2-(p-amidinophenyl)benzimidazole (I)
(Figure 1) was reported to show significant activity against several African
trypanosome strains and it was more active than the related benzothiazole
9
and benzoxazole analogues . However, since that report this system has
received little study. The closely related structural analogue 4’, 6-diamidino-
2-phenylindole, DAPI, (Figure 1), an extensively studied minor groove
DNA binder, was developed as a diamidino compound related to Berenil and
10
Stilbamidine, to be used as an antitrypanosomal agent . Recently, we have
demonstrated that modifications of DAPI could lead to more active
antiprotozoal agents 11. Based on the above findings, we report the
preparation of three types of benzimidazole diamidine compounds, by
replacing the phenyl moiety with heterocycles (5 a-c), and by substitution of
the benzimidazole NH of I with an NOH group (10) or by N-alkyl groups
(15 a-d).
2

Scheme 1 outlines the synthesis of the diamidines 5 a-c. The diamine 1
underwent oxidative cyclization by coupling with the aldehydes 2 a, b in the
7a
presence of 1, 4-benzoquinone in ethanol
or sodium bisulphite in
dimethylformamide 7b to give the benzimidazole derivatives 3 a, b in good
yields. The bromo compounds 3 a, b were converted to the corresponding
o
cyano derivatives 4 a, b by heating at 130-140 C overnight with copper
12
cyanide in dimethylformamide under a nitrogen atmosphere . Stirring the
dinitriles 4 a, b in ethanolic HCl according to Pinner methodology 7c, with
rigorous exclusion of water, afforded the corresponding bis-imidate ester
hydrochloride which was allowed to react with ethanol saturated with
ammonia gas to give the diamidines 5 a, b or to react with ethylene diamine
in ethanol to give the diimidazoline compound 5 c.
3

N
NH2
NH2
(a)
(b)
Br
OHC
N
H
X
X
NC
Br
NC
3 a,b
1
2 a,b
X
2 a
2 b
O
S
N
N
(c)
N
H
X
N
H
X
NC
CN
R
R
5 a-c
4 a,b
X
R
NH
5 a
O
NH2
NH
5 b
5 c
S
NH2
N
S
N
H
Scheme 1. Reagents and conditions
(a) i-1,4-benzoquinone, EtOH, or ii- Sodium bisulphite, DMF; (b)
CuCN, DMF; (c) i-HCl gas/EtOH, ii-NH3 gas/EtOH or NH2(CH2)2NH2/EtOH.
Scheme 2 gives the synthetic approach used to prepare the N-hydroxy
diamidino benzimidazole 10 starting with the activated chlorobenzene
derivative 6 which on reaction with the p-cyanobenzylamine 7 in dioxane in
the presence of potassium carbonate gives 8. The dicyano derivative 8 was
cyclized by heating in a sodium methoxide/ methanol solution to afford the
N-hydroxybenzimidazole 9. The diamidine 10 was obtained according to
Pinner methodology discussed before.
4

NH2
a
b
NC
Cl
NO2
NC
NH
NO2
+ NC
CN
8
6
7
N
N
N
c
NH
NH2
CN
HN
H2N
N
OH
NC
OH
10
9
Scheme 2. Reagents and conditions a) K2CO3, dioxane; b) MeOH, NaOMe; c) i-HCl gas/EtOH, ii-NH3 gas/EtOH.
Scheme 3 describes the synthesis of the diamidino benzimidazoles 15 a-d.
Alkylation of the activated chlorobenzene derivative 6 was achieved by
13
heating with an excess of the alkylamines to give 11 a-d in good yields .
The nitro compounds 11 a-d were hydrogenated at 50 psi using 10% Pd/C to
13
afford the diamines 12 a-d . The diamines 12 a-d underwent oxidative
cyclization by coupling with p-cyanobenzaldehyde in the presence of
7b
sodium bisulphite in dimethylformamide
to give the benzimidazole
derivatives 13 a-d in good yield. The bisnitrile 13a was converted to the
diamidine 15a using the Pinner approach described above. The dinitriles 13
b-d were allowed to react at room temperature for 24 h with a mixture of
hydroxylamine hydrochloride and potassium tert-butoxide in DMSO
solution to furnish the amidoximes 14 b-d in excellent yield. Acetate salts of
the diamidines 15 b-d were obtained from their respective diamidoximes 14
b-d, through the corresponding bis-O-acetoxyamidoximes, followed by
hydrogenation in glacial acetic acid/ethanol in the presence of 10% Pd/C 7b.
5

Table 1 contains the DNA binding affinities for the new diamidino
analogues of 5-amidino-2-(p-amidinophenyl)benzimidazole (I) as well as the
in vitro activity for these compounds against T. b. r. For comparative
purposes, data for I and DAPI are included. The thermal melting increase
Tm (Tm of complex – Tm of free DNA) is a rapid and reliable method for
14
ranking binding affinities for large numbers of aryldiamidines . The Tm
values for the complexes between poly (dA-dT) and the new analogues are
quite varied and ranges from 1.1 to 26oC. The thiophene compounds (5b
and 5c) exhibit high Tm values (25.2 and 26.0 oC) comparable to that of
DAPI and I. Interestingly, 5a, the furan isostere of 5b, gives a Tm value of
only 10.1 oC. This result is consistent with other DNA minor groove binders
which have shown stronger affinities for thiophene analogues than their
furan counterparts 15,16. The weaker DNA binding affinity of the furan
analogue 5a is likely due to the poorer fit with the minor groove as a result
of its greater curvature than that of 5b (see Figure 2).The N-alkyl
6

benzimidiazoles 15a-d all show relatively low Tm values ranging from 8.0
o
to 2.0 C. The drop in Tm values is consistent with the loss of the
benzimidazole NH which has been shown to play a key role in the binding
16
affinity of these type minor groove binders . Notably, the Tm values
roughly decrease with increasing size of the alkyl group. This interesting
result maybe be due to steric interactions between the alkyl groups and the
walls of the groove and/or may arise from the protrusion of the alkyl groups
out into solvent which may disrupt of waters of hydration in the minor
groove. Finally, the N-OH compound 10 has low binding (Tm 1.1 oC) to
poly (dA-dT). This result is counterintuitive as the NOH group provides
another potential H-bond donor. This result suggests that the N-OH faces
the floor of the groove and prevents the required close approach of the
diamidino groups to the floor of the groove and thereby eliminates important
H-bonding interactions between the amidine NHs and the thymine carbonyl
groups.
7

Figure 2. The furan 5 a and thiophene 5 b were energy minimized by geometry
optimization in the Spartan 10 software package with the molecular mechanics MMFF
force field. The benzimidazole group are quite constant in structure and are overlayed to
compare the structural changes at the furan and thiophene groups and amidines.
The in vitro activity against T. b. r. of the parent benzimidazole I is
slightly less than that of the related indole DAPI (IC50 values 31 and 18 nM,
respectively). Four of the eight new diamidines where highly active and
gave IC50 values less than 50 nM. The replacement of the phenyl group of I
with furan (5a) results in an increase in IC50 (48 nM), however replacement
with a thiophene (5b) provides a decrease in IC50 (19 nM). Replacement of
the amidines of 5b with imidazolines 5c results in a loss of activity (IC50 =
67 nM). Such a decline in activity on replacement of amidino groups with
cyclic amidines has been observed in other systems 7c. With the exception of
the N-methyl analogue 15a, which is the most active compound in this set
(IC50 = 15 nM and a selectivity index IC50-L6 / IC50-T.b.r of SI > 10,000),
all of the other N-alkyl compounds are less active than I. The N-cyclohexyl
analog 15d is significantly less active than I with an IC50 value of 529 nM
while compound 10 is inactive with an IC50 value of 8,433 nM. The two
most active compounds in vitro 5b and 15a, which also had reasonable
selectivity indices, were advanced to the stringent STB900 mouse model for
19
HAT . These compounds were evaluated at the low screening dose of 5
mg/kg. Neither compound showed significant in vivo activity. The in vivo
results for 5b and 15a are likely due to poor PK profiles.
Table 1. DNA biding affinities and biological data for the benzimidazole
analogues.
8

Cytotoxicity Selectivity
T.b.r
Tm
Compound
Number
index d
(nM)b
(Co)a
(M)c
NH
HN
48
DAPI
>27
18
31
0.86
26.3
N
H
NH2
NH2
NH
NH
N
H2N
848
I
24.7
N
H
NH2
N
NH
3229
O
5a
10.1
25.2
48
19
150.5
26.7
NH
H2N
NH2
NH
1405
1925
5b
5c
N
N
S
NH
26.0
8.0
67
15
64
129.2
>169
>207
N
HN
NH
NH
NH
N
H2N
>11266
>3234
15a
15b
N
NH2
Me
NH
NH
NH
N
N
H2N
H2N
5.5
NH2
NH
N
N
15c
2.5
31
>137
>4419
NH2
9

NH
NH
N
N
H2N
>306
>25
15d
10
2.0
1.1
529
>162
>210
NH2
N
N
NH
HN
H2N
8433
NH2
OH
Increase in thermal melting of poly(dA-dT)n in oC 17.
IC50 values obtained against the STIB900 strain of T. b. r. The IC50 values are the
a
b
average of two independent assays, the lower IC50 varied by less than 50%
from the higher IC50. 18.
18
c Cytotoxicity (IC50) was evaluated using cultured L6 rat myoblast cells . The IC50
values are the average of two independent assays, the lower IC50 varied by
less than 50% from the higher IC50.
d Selectivity index for T. b. r. expressed as the ratio: IC50 (L6)/IC50 (T.b.r.).
Several new analogues of I have been synthesized. The thiophene analogues
5b and 5c show DNA affinities comparable to I and DAPI. All the other
compounds showed lower binding affinities. The failure of the N-hydroxy
benzimidazole 10 to bind to the minor grove provides new insights regarding
placement of substituents which face the floor of the minor groove. The
thiophene analogue 5b and the N-methyl compound 15a show superior in
vitro antitrypanosomal activity compared to that of the parent, I, and despite
the low in vivo activity, this study provides excellent lead compounds for
optimization of PK properties.
Acknowledgments. This work was supported by an award from the Bill and
Melinda Gates Foundation through the Consortium for Parasitic Drug
Development (RB, WDW, DWB).
Supplementary data.
10

Supplementary data (complete characterizations of all the new compounds
associated with this article can be found, in the online version of this journal.
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13

Highlights:
1- New analogues of the antitrypanosomal agent DAPI were made.
2- Tm of some of the final amidines was superior than that of DAPI.
4- Compounds 5b and 15a showed superior antitrypanosomal activity
compared to the parent I.
14

Cytotoxicity Selectivity
T.b.r
Tm
Compound
Number
index d
(nM)b
(Co)a
(M)c
NH
HN
48
DAPI
>27
18
31
0.86
26.3
N
H
NH2
NH2
NH
NH
N
H2N
848
I
24.7
N
H
NH2
N
NH
3229
O
5a
10.1
25.2
48
19
150.5
26.7
NH
H2N
NH2
NH
1405
1925
5b
5c
N
N
S
NH
26.0
8.0
67
15
64
129.2
>169
>207
N
HN
NH
NH
NH
N
H2N
>11266
>3234
15a
15b
N
NH2
Me
NH
NH
NH
N
N
H2N
H2N
5.5
NH2
NH
N
N
15c
15d
2.5
2.0
31
>137
>162
>4419
>306
NH2
NH
NH
N
N
H2N
529
NH2
15

N
N
NH
HN
H2N
>25
10
1.1
8433
>210
NH2
OH
16

Graphical Abstract
NH
N
NH
N
N
H2N
HN
NH
N
H
X
NH2
R
NH2
H2N
X = O, S
R = CH3, CH2CH3, CH2CH2CH3, cyclohexyl
17